Bacteria rapidly develop resistance to antibiotic drugs within years of first clinical use. Antibiotic resistance can be acquired by horizontal gene transfer or result from persistence, in which a small fraction of cells in a population exhibits a non-inherited tolerance to antimicrobials. Since antimicrobial drug discovery is increasingly lagging behind the evolution of antibiotic resistance, there is a pressing need for new antibacterial therapies.
Bacterial infections are responsible for significant morbidity and mortality in clinical settings. Though the advent of antibiotics has reduced the impact of bacterial diseases on human health, the constant evolution of antibiotic resistance poses a serious challenge to the usefulness of today's antibiotic drugs. Infections that would have been easily cured by antibiotics in the past are now able to survive to a greater extent, resulting in sicker patients and longer hospitalizations. The economic impact of antibiotic-resistant infections is estimated to be between US $5 billion and US $24 billion per year in the United States alone. Resistance to antibiotic drugs develops and spreads rapidly, often within a few years of first clinical use. However, the drug pipelines of pharmaceutical companies have not kept pace with the evolution of antibiotic resistance.
Acquired antibiotic resistance results from mutations in antibacterial targets or from genes encoding conjugative proteins that pump antibiotics out of cells or inactivate antibiotics. Horizontal gene transfer, which can occur via transformation, conjugative plasmids, or conjugative transposons, is a major mechanism for the spread of antibiotic resistance genes. For example, Staphylococcus aureus became quickly resistant to sulpha drugs in the 1940s, penicillin in the 1950s, and methicillin in the 1980s. In 2002, staphylococci developed resistance to vancomycin, the only uniformly effective antibiotic against staphylococci, by receiving vancomycin-resistance genes via conjugation from co-infecting Enterococcus faecalis, which itself became completely resistant to vancomycin in nosocomial settings by 1988. Drugs such as ciprofloxacin that induce the SOS response can even promote the horizontal dissemination of antibiotic resistance genes by mobilizing genetic elements. For example, Streptococcus pneumoniae and Neisseria gonorrhoeae have also obtained resistance to antibiotics (Morens, et al., (2004) Nature 430: 242-249). Sub-inhibitory concentrations or incomplete treatment courses can present evolutionary pressures for the development of antibiotic resistance. Use of antibiotics outside of clinical settings, for example in livestock for the agricultural industry, has contributed to the emergence of resistant organisms such as methicillin-resistant staphylococci and is unlikely to abate due to economic reasons and modern farming practices. Resistance genes that develop in non-clinical settings may be subsequently transmitted to bacterial populations which infect humans, worsening the antibiotic resistance problem.
In addition to acquiring antibiotic-resistance genes, a small subpopulation of cells known as persisters can survive antibiotic treatment by entering a metabolically-dormant state. Persister cells do not typically carry genetic mutations but rather exhibit phenotypic resistance to antibiotics. In Escherichia coli, the fraction of a population that represents persister cells increases dramatically in late-exponential and stationary phases. Chromosomally-encoded toxins may be important contributors to the persister phenotype but the underlying mechanisms that control the stochastic persistence phenomena are not well understood. Persisters constitute a reservoir of latent cells that can begin to regrow once antibiotic treatment ceases and may be responsible for the increased antibiotic tolerance observed in bacterial biofilms. By surviving treatment, persisters may play an important role in the development of mutations or acquisition of genes that confer antibiotic resistance.
Several strategies have been proposed for controlling antibiotic resistant infections. New classes of antibiotics would improve the arsenal of drugs available to fight antibiotic-resistant bacteria but few are in pharmaceutical pipelines. Surveillance and containment measures have been instituted in government and hospitals so that problematic infections are rapidly detected and isolated but do not address the fundamental evolution of resistance. Cycling antibiotics is one method of controlling resistant organisms but is costly and may not be efficacious. Reducing the overprescribing of antibiotics has only moderately reduced antibiotic resistance. Efforts have been also made to lessen the use of antibiotics in farming but some use is inevitable.
Using bacteriophage to kill bacteria has been in practice since the early 20th century, particularly in Eastern Europe16, 17. Bacteriophage can be chosen to lyse and kill bacteria or can be modified to express lethal genes to cause cell death. However, bacteriophage which are directly lethal to their bacterial hosts can also produce phage-resistant bacteria in short amounts of time. In addition to the aforementioned approaches, novel methods for designing antimicrobial drugs are becoming more important to extending the lifespan of the antibiotic era. Combination therapy with different antibiotics or antibiotics with phage may enhance bacterial cell killing and thus reduce the incidence of antibiotic resistance, and reduce persisters. Unmodified filamentous bacteriophage have been shown to augment antibiotic efficacy. Systems biology analysis can be employed to identify pathways to target and followed by synthetic biology to devise methods to attack those pathways.
Bacterial biofilms are sources of contamination that are difficult to eliminate in a variety of industrial, environmental and clinical settings. Biofilms are polymer structures secreted by bacteria to protect bacteria from various environmental attacks, and thus result also in protection of the bacteria from disinfectants and antibiotics. Biofilms can be found on any environmental surface where sufficient moisture and nutrients are present. Bacterial biofilms are associated with many human and animal health and environmental problems. For instance, bacteria form biofilms on implanted medical devices, e.g., catheters, heart valves, joint replacements, and damaged tissue, such as the lungs of cystic fibrosis patients. Bacteria in biofilms are highly resistant to antibiotics and host defenses and consequently are persistent sources of infection.
Biofilms also contaminate surfaces such as water pipes and the like, and render also other industrial surfaces hard to disinfect. For example, catheters, in particular central venous catheters (CVCs), are one of the most frequently used tools for the treatment of patients with chronic or critical illnesses and are inserted in more than 20 million hospital patients in the USA each year. Their use is often severely compromised as a result of bacterial biofilm infection, which is associated with significant mortality and increased costs. Catheters are associated with infection by many biofilm-forming organisms such as Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Candida albicans, which frequently result in generalized blood stream infection. Approximately 250,000 cases of CVC-associated bloodstream infections occur in the US each year with an associated mortality of 12%-25% and an estimated cost of treatment per episode of approximately $25, 000. Treatment of CVC-associated infections with conventional antimicrobial agents alone is frequently unsuccessful due to the extremely high tolerance of biofilms to these agents. Once CVCs become infected the most effective treatment still involves removal of the catheter, where possible, and the treatment of any surrounding tissue or systemic infection using antimicrobial agents. This is a costly and risky procedure and re-infection can quickly occur upon replacement of the catheter.
Bacteriophages (often known simply as “phages”) are viruses that grow within bacteria. The name translates as “eaters of bacteria” and reflects the fact that as they grow, the majority of bacteriophages kill the bacterial host in order to release the next generation of bacteriophages. Naturally occurring bacteriophages are incapable of infecting anything other than specific strains of the target bacteria, undermining their potential for use as control agents.
Bacteriophages and their therapeutic uses have been the subject of much interest since they were first recognized early in the 20th century. Lytic bacteriophages are viruses that infect bacteria exclusively, replicate, disrupt bacterial metabolism and destroy the cell upon release of phage progeny in a process known as lysis. These bacteriophages have very effective antibacterial activity and in theory have several advantages over antibiotics. Most notably they replicate at the site of infection and are therefore available in abundance where they are most required; no serious or irreversible side effects of phage therapy have yet been described and selecting alternative phages against resistant bacteria is a relatively rapid process that can be carried out in days or weeks.
Bacteriophages (phages) prey on bacteria, infecting them, replicating and leaving the host, either by being shed non-lytically or lysing the host cell. The lytic property of bacteriophages led to them being discovered by Frederick Twort in 1915 (Twort Lancet 1915) and independently by Felix D'Herelle in 1917 (d'Herelle Comptes Rendus Hebdomadaires des Seances de L'academie des Sciences 1917), with D'Herelle recognizing the potential of these “bacteria-eaters” as a therapeutic modality. Bacteriophage therapy was successfully used to combat bacterial infections in Africa and India against cholera and to disinfect water wells. Historically, bacteriophage therapy predates the widespread use of antibiotics, but due to the advent of broad-spectrum antibiotics in the western world, this form of anti-infective treatment has not been pursued. Most, if not all, bacteriophage therapy is performed in the former Soviet Republic states due to the continued development and refinement of bacteriophage therapy approaches during the cold war (Stone Science 2002; Deresinski., Clin Infect. Diseases, 2009).
However, western practitioners have shied away from harnessing phage therapy, citing two primary concerns: i) the exquisite specificity of bacteriophages which means they can't be used like broad-spectrum antibiotics and necessitate a shift in clinical treatment protocol towards combination treatments, and ii) the quick development of phage resistance by strains of bacteria while they are being treated (Skurnik and Strauch., Int. J. Med. Microbiol. 2006).
Bacteriophages have been used in the past for treatment of plant diseases, such as fireblight as described in U.S. Pat. No. 4,678,750. Also, Bacteriophages have been used to destroy biofilms (e.g., U.S. Pat. No. 6,699,701). In addition, systems using natural bacteriophages that encode biofilm-destroying enzymes in general have been described. Art also provides a number of examples of lytic enzymes encoded by bacteriophages that have been used as enzyme dispersion to destroy bacteria (U.S. Pat. No. 6,335,012 and U.S. Patent Application Publication No. 2005/0004030).
The Eastern European research and clinical trials, particularly in treating human diseases, such as intestinal infections, has apparently concentrated on use of naturally occurring phages and their combined uses (Lorch, A. (1999), “Bacteriophages: An alternative to antibiotics?” Biotechnology and Development Monitor, No. 39, p. 14-17). For example, non-engineered bacteriophages have been used as carriers to deliver antibiotics (such as chloroamphenicol) (Yacoby et al., Antimicrobial agents and chemotherapy, 2006; 50; 2087-2097). Non-engineered bacteriophages have also had aminoglycosides antibiotics, such as chloroamphenicol, attached to the outside of filamentous non-engineered bacteriophage (Yacoby et al., Antimicrobial agents and chemotherapy, 2007; 51; 2156-2163). Non-engineered filamentous Pf3 bacteriophages have been reported to be administered with low concentration of gentamicin, where neither the filamentous Pf3 nor the gentamicin could eliminate the bacterial infection alone (Hagens et al, Microb. Drug resistance, 2006; 12; 164-8). Simultaneous administration of non-engineered bacteriophages and the antibiotic enrofloxacin have been reported, however the use of the antibiotic alone was reported to be more effective than the combination of the antibiotic and bacteriophage (see Table 1 in Huff et al., 2004; Poltry Sci, 83; 1994-1947).
Although there have been some reports of engineered bacteriophages, these have not been widely developed. For example, engineered M13 non-lytic bacteriophage that carry lethal cell death genes Gef and ChpBK. (Westwater et al., 2003, Antimicrobial agents and chemotherapy, 47; 1301-1307) have been reported.
Constant evolutionary pressure will ensure that antibiotic resistance bacteria will continue to grow in number. The lack of new antibacterial agents being developed in the last 25-30 years certainly bodes poorly for the future of the antibiotic era (Wise, R (2004) J Antimicrob Chemother 54: 306-310). As a result, there has been growing interest in phage therapy due to the advent of a greater number of antibiotic-resistant strains of bacteria (Merril, Scholl et al. Nature reviews Drug discovery). The specificity of bacteriophages is dependent on the specificity of the interaction between the tail fibers of the bacteriophage and the recognized domain(s) of the bacteria for which the bacteriophage exhibits tropism (Liu, Deora et al. Science 2002; Dai, Hodes et al. Proc Natl Acad Sci USA 2010). It is generally held to be true that bacteriophages are not able to infect more than a handful of closely related sub-species of bacteria, much less bacteria from different strains (Lederberg et al., Proc Natl Acad Sci USA 1996).
However, recent work has shown that the specificity of bacteriophages in common usage might be an artifact of historical isolation procedures used, which bias the isolation towards the most infective bacteriophage, with the greatest burst size (Rabinovitch, Hadas et al. J Bacteriol 1999). Specifically, it is feasible to change the protocols for isolation of phage to grow a desired phage on multiple hosts over multiple rounds. This ensures a broader selectivity of the phage throughout the passage from the initial input material. The isolation of bacteriophages with multiple specificities without great difficulty and only minor changes in the isolation protocols speaks to the enormous reservoir of variability in nature, and makes the use of single or low numbers of combinations of bacteriophages in an anti-infective setting more feasible (Jensen, Schrader et al. Appl Environ Microbiol 1998).
Similarly, it is considered a by-product of millions of years of co-evolution in the bacteria-bacteriophage predator-prey system that the prey (bacteria) have evolved the ability to quickly shift to a more resistant form in response to predation by bacteriophages. This prevents complete elimination of the prey species by the predating species, which would also result in a catastrophic extinction of the predating species. This fact has previously made phage therapy a less desirable alternative to antibiotic therapy.
Because antibiotic resistance in treating bacterial infections and biofilms poses a significant hurdle to eliminating or controlling or inhibiting bacteria and biofilms with conventional antimicrobial drugs, new anti-biofilm strategies, such as phage therapy, should be explored. Novel synthetic biology technologies are needed to enable the engineering of natural phage with biofilm-degrading enzymes to produce libraries of enzymatically-active phage, which can complement efforts to screen for new biofilm-degrading bacteriophages in the environment.
Thus, new methods for combating bacterial infections are needed in order to prolong the antibiotic age. For example, bacteriophage therapy or synthetic antibacterial peptides have been proposed as potential solutions (Loose et al., (2006) Nature 443: 867-869; Curtin, et al., (2006) Antimicrob Agents Chemother 50: 1268-1275).